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Dermorphin - an overview

Chapters and Articles You might find these chapters and articles relevant to this topic. 2004, ToxiconWudayagiri Rajendra, ... Kandiah Jeyaseelan Extensive in vivo and in vitro investigations have shown that dermorphins are highly potent, selective and subtype specific μ-opiate receptor agonists. For example, [Lys7]- dermorphin shows high affinity for μ1-receptors, whereas [Trp4, Asn7]-dermorphin binds preferentially to the low affinity μ2-receptors (Negri et al., 1992). Like μ-opiate agonists dermorphins also produce anti-nociception, catalepsy, rigidity and sedation. Intrathecal administration of dermorphins in humans has shown 100 times greater potency than morphine (Negri et al., 1992). The synthetic peptides [Arg7]-dermorphins, [Hyp6 Lys7]-dermorphin and [Lys7, 8]-dermorphins and some glycosylated analogs also showed anti-nociceptive activity with higher permeability propensity through Blood-Brain-Barrier unlike the naturally occurring dermorphins (Negri et al., 1998). Read full a

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Chapters and Articles

You might find these chapters and articles relevant to this topic.

2004, ToxiconWudayagiri Rajendra, ... Kandiah Jeyaseelan

Extensive in vivo and in vitro investigations have shown that dermorphins are highly potent, selective and subtype specific μ-opiate receptor agonists. For example, [Lys7]- dermorphin shows high affinity for μ1-receptors, whereas [Trp4, Asn7]-dermorphin binds preferentially to the low affinity μ2-receptors (Negri et al., 1992). Like μ-opiate agonists dermorphins also produce anti-nociception, catalepsy, rigidity and sedation. Intrathecal administration of dermorphins in humans has shown 100 times greater potency than morphine (Negri et al., 1992). The synthetic peptides [Arg7]-dermorphins, [Hyp6 Lys7]-dermorphin and [Lys7, 8]-dermorphins and some glycosylated analogs also showed anti-nociceptive activity with higher permeability propensity through Blood-Brain-Barrier unlike the naturally occurring dermorphins (Negri et al., 1998).

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URL: https://www.sciencedirect.com/science/article/pii/S0041010104001862

2004, ToxiconWudayagiri Rajendra, ... Kandiah Jeyaseelan

Several lines of evidence demonstrated that μ- and δ- agonists of opioid receptors localized in dorsal horn neurons of spinal cord produce anti-nociception. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and related heptapeptide [Hyp6]-dermorphin isolated from the frog skin of Phyllomedusa sp, show higher affinity for μ-opioid receptors (Broccardo et al., 1981; Montecucchi et al., 1981). Several peptides belonging to the dermorphin family, have been isolated from frog skin (Barra et al., 1994; Naim et al., 1993; Mor et al., 1989; Broccardo et al., 1981; Improta and Broccardo, 1992).

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URL: https://www.sciencedirect.com/science/article/pii/S0041010104001862

The dermorphin-derived tetrapeptide [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2), containing 2′,6′-dimethyltyrosine (Dmt) in the 1-position of the peptide sequence, is a highly potent μ opioid agonist (Schiller et al. 2000). Its μ receptor binding affinity (Kiμ = 0.143 nM) is about 7-fold higher than that of morphine and it has high μ receptor binding selectivity [Ki ratio (μ/δ/κ) = 1/14700/156]. When incubated in sheep blood, [Dmt1]DALDA was found to be highly stable against enzymatic degradation, as was to be expected on the basis of its structural characteristics (Szeto et al. 2001). This compound showed extraordinary potency (3000 times more potent than morphine) in the rat tail-flick assay with intrathecal (i.th.) administration (Shimoyama et al. 2001). The observation that [Dmt1]DALDA has only 7-fold higher μ receptor binding affinity than morphine suggested that additional mechanisms may be implicated in producing the very potent analgesic effect of this compound at the spinal level. Indeed, [Dmt1]DALDA was shown to inhibit norepinephrine uptake in rat spinal cord synaptosomes with an IC50 of 4.1 μM (Shimoyama et al. 2001). Due to the latter effect, norepinephrine levels are increased and activation of both α2-adrenergic receptors and μ opioid receptors takes place which is known to produce an enhanced analgesic effect in a synergistic manner (Reimann et al. 1999). This interpretation is supported by the observation that the antinociceptive response to [Dmt1]DALDA is attenuated by the α2-adrenergic antagonist yohimbine (Shimoyama et al. 2001). Furthermore, it was shown that the i.th. antinociceptive effect of [Dmt1]DALDA was decreased after pretreatment (i.th.) with the κ opioid antagonist norBNI, or with antiserum against dynorphin A(1–17) or [Met5]enkephalin (Szeto et al. 2003). These results were interpreted to indicate that this peptide also acts as a releaser of dynorphin- and [Met5]enkephalin-like substances that act at κ and δ receptors to contribute to the extraordinary antinociceptive potency of [Dmt1]DALDA. Thus, [Dmt1]DALDA is a spinal analgesic with triple action: μ opioid receptor activation, inhibition of norepinephrine uptake and release of endogenous opioid peptides. In the rat tail-flick test, this peptide produced an antinociceptive effect that lasted four times longer than that of morphine when both compounds were administered i.th. at equipotent doses (3 × ED50) (Shimoyama et al. 2001). Both its enzyme resistance and its slow clearance from the spinal cord due to its high positive charge may be the reason for the long duration of action of [Dmt1]DALDA. Another advantage of this compound is that it has a very low propensity to produce respiratory depression (Shimoyama et al. 2001). For all these reasons, [Dmt1]DALDA may be considered as a promising drug candidate for use in spinal analgesia. The only drawback of this compound is that it produced quite profound tolerance after chronic i.th. administration (Zhao et al. 2002; Ben et al. 2004). [Dmt1]DALDA was also 100–200-fold more potent than morphine in the mouse tail-flick test when given by the intracerebroventricular (i.c.v.) route (Neilan et al. 2001; Zhao et al. 2002).

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URL: https://www.sciencedirect.com/science/article/pii/S0024320509001015

The Dermaseptin Superfamily: A Library of Structurally and Functionally Disparate Peptides Originating from a Single Gene Family

Hylidae (tree frogs) are one of the largest families of frogs. There are about 51 genera that are arranged in three subfamilies: Pelodryadinae, Phyllomedusinae, and Hylinae. The first member of the dermaseptin superfamily, named dermaseptin-S1, was isolated from an extract of dried pieces of skin of Phyllomedusa sauvagii (subfamily: Phyllomedusinae) in the early 1990s.26 This 34-residue peptide has lytic activity against Gram-positive and Gram-negative bacteria, yeast, and protozoa, without harmful effects against mammalian cells. It was the first gene-encoded eukaryotic peptide to show lethal effect against Leishmania promastigotes16 and filamentous fungi that are responsible for opportunistic infections resulting from the immunodeficiency syndrome or the use of immunosuppressive agents. This discovery was followed by the isolation of adenoregulin (also named dermaseptin-B2) from P. bicolor skin, a peptide that interacts with the adenosine receptor,11 and dermaseptin-B1.27 These 2 peptides were thought to be unrelated until attempts to clone their precursor polypeptides revealed the presence of a common preproregion and 5′- and 3′-UTRs as well as extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins from Phyllomedusa skin.2,3 Since then, additional members of the dermaseptin superfamily were rapidly identified in various species of South American Hylidae frogs and grouped to form distinct families on the basis of their structural characteristics: (1) the dermaseptins (stricto sensu) from Agalychnis annae, A. callidryas, A. dacnicolor, A. lemur, A. spurrelli (litodryas), Phasmahyla jandai, Phyllomedusa bicolor, P. burmeisteri, P. distincta, P. hypochondrialis, P. oreades, P. sauvagii, P. tarsius, P. tomopterna, and P. trinitatis (Table 1). Most of these peptides share a signature pattern consisting of a conserved Trp residue at position 3 and an AA(A/G)KAAL(G/N)A consensus motif in the midregion; (2) the dermatoxins from Agalychnis annae, A. dacnicolor, Phasmahyla jandaia, Phyllomedusa bicolor, P. sauvagii, and P. tarsius (Table 2); (3) the phylloxins from Phyllomedusa bicolor and P. sauvagii (Table 3); (4) the phylloseptins from Agalychnis lemur, Phasmahyla jandaia, Phyllomedusa bicolor, P. burmeisteri, P. hypochondrialis, P. palliata, P. sauvagii, P. tarsius, and P. tomopterna (Table 4); (5) the plasticins from Agalychnis dacnicolor, A. annae A. callidryas, Phyllomedusa bicolor, and P. sauvagii, which are rich in Gly and Leu residues arranged in regular 5-mer motifs GXXXG (where X is any amino acid residue) (Table 5); (6) the raniseptins from Hypsiboas raniceps (Table 6); (7) the caerin-related peptides from Agalychnis annae and A. callidryas (Table 7); and (8) the orphan peptides from Agalychnis dacnicolor, Phyllomedusa hypochondrialis, and P. sauvagii, which do not resemble any members of the other peptide families (Table 7). As shown in Tables 1–7, different species of hylid frogs are equipped with different sets of peptides belonging to different families. Orthologous peptides and paralogous peptides have different amino acid sequences in different species and most often display differential activity against a range of microorganisms. This impressive divergence between and within species is such that the evolutionary relationships of these peptides would never have been apparent without the strong conservation of their biosynthetic precursor preproregions (Section 2). Australian hylid frogs belonging to the Pelodryadinae subfamily also produce in their skin several families of antimicrobial peptides (caerins, fallaxidins, frenatins, aureins) (see the chapter by Bowie and Tyler, in this Section of the book) that have no structural similarity with South American hylid peptides, but the signal peptide and intervening sequences of the precursors of the caerins, fallaxidins, frenatins, and aureins are highly similar to the corresponding regions of the precursors of the South American hylid antimicrobial peptides (Table 9). Similarly, numerous antimicrobial peptides from Eurasian and North American frogs (families: Ranidae and Dicroglossidae) that belong to the brevinin, esculentin, nigrocin, palustrin, ranacyclin, ranalexin, ranatuerin, amolopin, temporin, and limnonectin34 families have precursors whose preproregions strikingly resemble those of preprodermaseptins (Table 9) (see the chapter by Conlon in this Section). Frog skin secretions are also a rich source of biologically active neuropeptides and hormones that are very similar to mammalian peptides that are produced in the brain and the gastrointestinal tract. Most interestingly, preprodermaseptin-encoded peptides also include D-amino acid-containing opioid peptides belonging to the dermorphin/deltorphin family (see the chapter by Negri and Melchiorri in this Section), bradykinin-related peptides, the myoactive peptides hyposins and tryptophyllins, and Bowman–Birk-like trypsin inhibitors32 (Tables 8 and 9). The discovery of the considerable extent of sequence identity between the neuropeptide and antimicrobial peptide precursors is unprecedented. Thus, although the two groups of peptides are genetically related and belong to the same gene superfamily, they have strongly diverged to yield families of peptides that are both structurally and functionally distinct.

TABLE 1. The Dermaseptin (stricto sensu) family. Amino acid Sequences of Dermaseptins stricto sensu from South American Hylid Frogs (Subfamily Phyllomedusinae). X = Leu or Ile. a = C-Terminal Amidation. Names are Proposed According to the Frog Species Names Set Out in AMPHIBIAN Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs 4, 31. The Original Nomenclatures are in Brackets. ARP = adenoregulin-related peptide; DRS, DS = dermaseptin; DRP = dermaseptin-related peptide; DD = dermadistinctin; DRG = dermaseptin gene related. Accession number: http://www.uniprot.org/uniprot

Genus, species Dermaseptin Abbreviation Sequence Accession number/Ref. Agalychnis A. annae A3 DRS-A3(DRP-AA-3-1) SLWSKIKEMAATAGKAALNAVTGMVNQa O93223 A4 DRS-A4(DRP-AA-3-3) GMFTNMLKGIGKLAGQAALGAVKTLAa O93224 A5 DRS-A5(DRP-AA-3-6) GMWSTIRNVGKSAAKAANLPAKAALGAISEAVa O93226 A. callidryas C1 DRS-C1(ARP-AC1) GMWSKIKEAGKAAAKAAAKAAGKAALDVVSGAIa B6HY15 C2 DRS-C2(DRP-AC1) SLLSTLGNMAKAAGRAALNAITGLVNQa B6YH16 C3 DRS-C3(DRP-AC3) SVLSTITDMAKAAGRAALNAITGLVNQa Q800S0 A. dacnicolor (Pachymedusa dacnicolor) DA2 DRS-DA2(DRP-PD-2-2) ALWKTLLKKVGKVAGKAVLNAVTNMANQNEQ O93452 DA3 DRS-DA3(DRP-PD-3-3) GMWSKIKNAGKAAAKASKKAAGKAALGAVSEALa O93453 DA4 DRS-DA4 GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM 7 DA5 DRS-DA5 GMWGKIKSTAKEAAKAAGKAALNAVSEAXa 25 DA6 DRS-DA6 GVWGIAKIAGKVLGNILPHVFSSNQSa 25 DA7 DRS-DA7 GXWNSXKEGGVGXAKQNVVVTGMVNQa 25 DA8 DRS-DA8 GLWNSIKDMAAAAGRAALNAVTGMVNQa 25 A. lemur L1 DRS-L1 GLWSKIKEAAKAAGKAALNAVTGLVNQGDQPS 10 (Hylomantis lemur) A. spurrelli LI1 DRS-LI1(Peak-2.9) AVWKDFLKNIGKAAGKAVLNSVTDMVNE 24 (A. litodryas) Phasmahyla P. jandaia J1 DRS-J1 GLWKNMLSGIGKLAGQAALGAVKTLVa P86635 J2 DRS-J2 GLWKNMLSGIGKLAGEAALGAVKTLVa P86636 J3 DRS-J3 ALWKNMLSGIGKLAGQAALGAVKTLVa P86637 J4 DRS-J4 ALWKDMLSGIGKLAGQAALGAVKTLVa P86638 J5 DRS-J5 GLWSKIKEAGKAAVKAAGKAALGAVANSVa P86683 J6 DRS-J6 GLWSKIKEAGKAAVKAAGKAALGAVADSVa P86684 J7 DRS-J7 GLWSKIKAAGKEAAKAAAKAAGKAALNAVSEAVa P86639 J8 DRS-J8 GLWKSLLKNVGKAAGKAALNAVTDMVNQA P86640 J9 DRS-J9 GLWKSLLKNVGKAAGKAALNAVTDMVNQS P86641 J10 DRS-J10 ALWKSLLKGAGQLVGGVVQHFMGSQGQPES P86642 Phyllomedusa P. bicolor B1 DRS-B1 AMWKDVLKKIGTVALHAGKAALGAVADTISQa P80282 B2 DRS-B2(adenoregulin) GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVa P31107 B3 DRS-B3 ALWKNMLKGIGKLAGQAALGAVKTLVGAE P81485 B4 DRS-B4 ALWKDILKNVGKAAGKAVLNTVTDMVNQa P81486 B5 DRS-B5 GLWNKIKEAAKSAGKAALGFVNEMVa P81487 B6 DRS-B6 ALWKDILKNAGKAALNEINQLVNQa P81490 B7 DRS-B7(DRG1) GLWSNIKTAGKEAAKAALKAAGKAALGAVTDAVa Q90ZK3 B8 DRS-B8(DRG2) GLWSKIKEAGKAVLTAAGKAALGAVSDAVa Q90ZK5 B9 DRS-B9(DRG3) ALWKTIIKGAGKMIGSLAKNLLGSQAQPESEQ P81488 P. burmeisteri BU1 DRS-BU1(DS III-like) ALWKNMLKGIGKLAGKAALGAVK P86281 BU2 DRS-BU2(DRSDI4-like) ALWKNMLKGIGKLAGQAALGAVKTLVGA P86279 BU3 DRS-BU3(DS VIII-like) ALWKTMLKKLGTVALHAGKAALGAAADTISQGA P86280 P. distincta DI1 DRS-DI1(DD K) GLWSKIKAAGKEAAKAAAKAAGKAALNAVSEAV P83638 DI2 DRS-DI2(DD L) ALWKTLLKNVGKAAGKAALNAVTDMVNQ P83639 DI3 DRS-DI3(DD M) ALWKTMLKKLGTMALHAGKAAFGAAADTISQ P83640 DI4 DRS-DI4(DD Q1) ALWKNMLKGIGKLAGQAALGAVKTLVGAES P83641 DI5 DRS-DI5(DD Q2) GLWSKIKEAAKTAGLMAMGFVNDMV P83642 P. hypochondrialis/azurea H1 DRS-H1(DShypo 02) GLWKSLLKNVGVAAGKAALNAVTDMVNQa P84597 H2 DRS-H2 ALWKSLLKNVGVAAGKAALNAVTDMVNQa Q0VZ36 H3 DRS-H3 GLWSTIKNVAAAAGKAALGALa Q17UY8 H4 DRS-H4(DShypo 01) GLWSTIKNVGKEAAIAAGKAALGALa Q1EJP5 H5 DRS-H5 GLWSTIKNVGKEAAIAAGKAVLGSLa Q1EJP4 H6 DRS-H6 GLWSTIKQKGKEAAIAAAKAAGQAALGALa P84936 H7 DRS-H7 GLWSKIKDVAAAAGKAALGAVNEALa P84937 H8 DRS-H8(DShypo03) ALWKDVLKKIGTVALHAGKAAFGAAADTISQGGS P84598 H9 DRS-H9(DShypo 04) GLWSTIKQKGKEAAIAAAKAAGKAVLNAASEALa P84599 H10 DRS-H10(DS 01) GLWSTIKQKGKEAAIAAAKAAGQAALGALa P84600 H11 DRS-H11(DShypo 06) GLWSTIKQKGKEAAIAAAKAAGQAVLNSASEALa P84601 H12 DRS-H12(DShypo 07) GLWSTIKQKGKEAAIAAAKAAGQAALNAASEALa P84880 P. oreades O1 DRS-O1(DS 01) GLWSTIKQKGKEAAIAAAKAAGQAALGALa P83637 P. sauvagii S1 DRS-S1(dermaseptin) ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ P24302 S2 DRS-S2 ALWFTMLKKLGTMALHAGKAALGAAANTISQGTQ P80278 S3 DRS-S3 ALWKNMLKGIGKLAGKAALGAVKKLVGAES P80279 S4 DRS-S4 ALWMTLLKKVLKAAAKALNAVLVGANA P80280 S5 DRS-S5 GLWSKIKTAGKSVKAAAKAAVKAVTNAV P80281 S6 DRS-S6 GLWSKIKTAGKEAAKAAAKAAGKAALNAVSEAIa Q7T3K9 S7 DRS-S7 GLWKSLLKNVGKAAGKAALNAVTDMVNQa Q7T3K8 S8 DRS-S8 ALWKTMLKKLGTVALHAGKAALGAAADTISQa Q7T3K7 S11 DRS-S11 ALWKTLLKGAGKVFGHVAKQFLGSQGQPES Q1EN13 S12 DRS-S12 GLWSKIKEAAKTAGKMAMGFVNDMVa Q1EN12 S13 DRS-S13 GLRSKIKEAAKTAGKMALGFVNDMAa Q1EN11 P. tarsius TA1 DRS-TA1(DStar 01) GLWSKIKETGKEAAKAAGKAALNKIAEAVa P84921 TA2 DRS-TA2(DStar 02) ALWKDILKNVGKAAGKAVLNTVTDMVNQa P84922 TA3 DRS-TA3(DStar 03) GLFKTLIKGAGKMLGHVAKQFLGSQGQPESa P84923 TA4 DRS-TA4(DStar 04) ALWKDILKNAGKAALNEINQIVQa P84924 TA5 DRS-TA5(DStar 05) GLWSKIKEAAKTAGKAAMGFVNEMVa P84925 TA6 DRS-TA6(DStar 06) ALWKNMLKGIGKLAGQAALGAVKTLVGA P84926 TA7 DRS-TA7(DStar 07) ALWKDVLKKIGTVALHAGKAALGAVADTISQ P84927 P. tomopterna TO1 DRS-TO1(DStomo01) ALWKDLLKNVGIAAGKAVLNKVTDMVNQa P85523 P. trinitatis TR1 DRS-TR1(DRS-B4) ALWKDILKNVGKAAGKAVLNTVTDMVNQa P81486

TABLE 2. The Dermatoxin family. Amino acid Sequences of Dermatoxins from South American Hylid Frogs (Subfamily Phyllomedusinae). a = C-Terminal Amidation. Names are Proposed According to the Frog Species Names Set out in Amphibian Species of the World (http://research.amnh.org/vz/ Herpetology/Amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. DRT = dermatoxin; DS = dermaseptin; DRP = dermaseptin-related peptide

Genus, species Dermatoxin Abbreviation Sequence Accession number Agalychnis A. annae A1 DRT-A1(DRP-AA-1-1) SLGSFMKGVGKGLATVGKIVADQFGKLLEAGQa O93221 A. dacnicolor (Pachymedusa dacnicolor) DA1 DRT-DA1(DRP-PD-1-5) SLGSFMKGVGKGLATVGKIVADQFGKLLEAGKG O93451 Phasmahyla P. jandaia J1 DRT-J1 SLGGFLKGVGKALAGVGKVVADQFGNLLQAGQa P86621 J2 DRT-J2 SLGGFLKGVGKALAGVGKMVADQFGNLLQAGQa P86622 J3 DRT-J3 SLGGFLKGVGKVLAGVGKVVADQFGNLLEAGQa P86623 Phyllomedusa P. bicolor B1 DRT-B1(dermatoxin) SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQa Q9PT75 P. sauvagii S1 DRT-S1(DRT-S) ALGTLLKGVGSAVATVGKMVADQFGKLLQAa Q5DVA5 P. tarsius TA1 DRT-TA1(DStar 08) SLRGFLKGVGTALAGVGKVVADQFDKLLQAGQa P84928

TABLE 3. The Phylloxin family. Amino Acid Sequences of Phylloxins from South American Hylid Frogs (Subfamily Phyllomedusinae). a = C-Terminal Amidation. Names are Proposed According to the Frog Species Names Set out in Amphibian Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. PLX = phylloxin

Genus, species Phylloxin Abbreviation Sequence Accession number Phyllomedusa P. bicolor B1 PLX-B1 (phylloxin) GWMSKIASGIGTFLSGMQQa P81565 P. sauvagii S1 PLX-S1 (phylloxin S) GWMSKIASGIGTFLSGVQQa Q5DVA6

TABLE 4. The Phylloseptin family. Amino Acid Sequences of Phylloseptins from South American Hylid frogs (Subfamily Phyllomedusinae). a = C-Terminal Amidation. Names are Proposed According to the Frog Species Names Set Out in Amphibian Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. PLS, PS, PSN = phylloseptin; PBN1 = Phyllomedusa bicolor new 1

Genus, species Phylloseptin Abbreviation Sequence Accession number/Ref. Agalychnis A. lemur (Hylomantis lemur) L1 PLS-L1 LLGMIPLAISAISALSKLa 10 L2 PLS-L2 FLSLIPHVISALSSLa 1 Phasmahyla P. jandaia J1 PLS-J1 FLSLIPHAINAISAIANHFa P86614 J2 PLS-J2 FLSLIPHAINAISAIADHFa P86615 J3 PLS-J3 FLSLIPHAINAISAIANHLa P86616 J4 PLS-J4 FLSLIPHAINAISAIAHHLa P86617 J5 PLS-J5 FLSLIPHAISAISAIAHHLa P86618 J6 PLS-J6 FLSLIPHAISAISAIANHLa P86619 J7 PLS-J7 FLSLIPHAISAISAIADHLa P86620 Phyllomedusa P. bicolor B1 PLS-B1(PBN1) FLSLIPHIVSGVAALAKHLa Q800R3 P. burmeisteri BU1 PLS-BU1(BU-1) FLISIPYSASIGGTATLTGTAa P86282 BU2 PLS-BU2(BU-2) FLLSLPHLASGLASLVLSKa P86283 P. hypochondrialis/azurea H1 PLS-H1(PS-1) FLSLIPHAINAVSAIAKHNa P84566 H2 PLS-H2(PS-2) FLSLIPHAINAVSTLVHHFa P84567 H3 PLS-H3(PS-3) FLSLIPHAINAVSALANHGa P84568 H4 PLS-H4(PS-6) SLIPHAINAVSAIAKHFa P84571 H5 PLS-H5(PS-7) FLSLIPHAINAVSAIAKHFa P85882 H6 PLS-H6(PS-8) FLSLIPTAINAVSALAKHFa Q0VZ41 H7 PLS-H7(PS-9) FLGLLPSIVSGAVSLVKKLa Q0VZ38 H8 PLS-H8(PS-10) FLSLLPSLVSGAVSLVKKLa Q0VZ39 H9 PLS-H9(PS-11) FLSLLPSLVSGAVSLVKILa Q0VZ40 H10 PLS-H10(PS-12) FLSLLPSIVSGAVSLAKKLa Q17UY9 H11 PLS-H11(PS-13) FLSLIPHAINAVGVHAKHFa P84938 H12 PLS-H12(PS-14) FLSLIPAAISAVSALADHFa P84939 H13 PLS-H13(PS-15) LLSLVPHAINAVSAIAKHFa Q0VKG9 P. oreades O1 PLS-O1(PS-4) FLSLIPHAINAVSTLVHHSa P84569 O2 PLS-O2(PS-5) FLSLIPHAINAVSAIAKHSa P84570 P. palliata P1 PLS-P1 FLSLIPHAINAVSAIAKHFa P86710 P. sauvagii S1 PLS-S1(PSN-1) FLSLIPHIVSGVASIAKHFa E3PQH3 P. tarsius TA1 PLS-TA1(PStar 01) FLSLIPKIAGGIASLVKNLa P84929 TA2 PLS-TA2(PStar 02) FLSLIPHIATGIAALAKHLa P84930 TA3 PLS-TA3(PStar 03) FFSMIPKIATGIASLVKNLa P84931 P. tomopterna TO1 PLS-TO1(PLS-8) FLSLIPHAINAVSALAKHFa P85447

TABLE 5. The Plasticin family. Amino acid Sequences of Plasticins from South American Hylid Frogs (Subfamily Phyllomedusinae). a = C-Terminal Amidation. Names are Proposed According to the Frog Species Names Set out in Amphibian Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. DRP = dermaseptin-related peptide; PBN2 = Phyllomedusa bicolor new 2; DRS = dermaseptin; PTC = plasticin

Genus, species Plasticin Abbreviation Sequence Accession number Agalychnis A. annae A1 PTC-A1(DRP-AA-2-5) GLVSGLLNTAGGLLGDLLGSLGSLSGa O93222 A. callidryas C1 PTC-C1(DRP-AC1) GLLSGILNTAGGLLGNLIGSLSNa Q800S2 C2 PTC-C2(DRP-AC2) GLLSGILNSAGGLLGNLIGSLSNa Q800S1 A. dacnicolor (Pachymedusa dacnicolor) DA1 PTC-DA2(DRP-PD-3-6) GVVTDLLNTAGGLLGNLVGSLSGa O93454 Phyllomedusa P. bicolor B1 PTC-B1(PBN2) GLVTSLIKGAGKLLGGLFGSVTGa Q800R4 P. sauvagii S1 PTC-S1(DRS-S10) GLVSDLLSTVTGLLGNLGGGGLKKI Q1EN14

TABLE 6. The Raniseptin family. Amino Acid Sequences of Raniseptins from South American Hylid Frogs (Subfamily Hylinae). Names are Proposed According to the Frog Species Names Set out in Amphibian Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. RSP = raniseptin

Genus, species Raniseptin Abbreviation Sequence Accession number Hypsiboas H. raniceps (Hyla roeschmanni) R1 RSP-R1(Rsp-1) AWLDKLKSLGKVVGKVALGVAQNYLNPQQ P86037 R2 RSP-R2(Rsp-2) AWLDKLKSLGKVVGKVAIGVAQHYLNPQQ P86185 R3 RSP-R3(Rsp-3) AWLDKLKSIGKVVGKVAIGVAKNLLNPQ P86038 R4 RSP-R4(Rsp-4) AWLDKLKSLGKVVGKVGLGVVQNYLNPRQ P86186 R5 RSP-R5(Rsp-5) AWLDKLKNLGKVVGKVALGVVQNYLNPRQ P86187 R6 RSP-R6(Rsp-6) ALLDKLKSLGKVVGKVALGVVQNYLNPRQ P86039 R7 RSP-R7(Rsp-7) ALLDKLKSLGKVVGKVALGVAQHYLNPQQ P86040 R8 RSP-R8(Rsp-8) ALLDKLKSLGKVVGKVAIGVAQHYLNPQQ P86188 R9 RSP-R9(Rsp-9) ALLDKLKSLGKVVGKVAIGVAQHYLNPQ P86189

TABLE 7. The Caerin-Related Peptide Family and Orphan Antimicrobial Peptides. Amino Acid Sequences of Caerin-Related Peptides and Orphan Peptides from South American Hylid Frogs (Subfamily Phyllomedusinae). Names are Proposed According to the Frog Species Names Set out in Amphibian Species of the World (http://research.amnh.org/vz/herpetology/amphibia) and Refs. 4, 31. The Original Nomenclatures are in Brackets. CRP = caerin-related peptide; DRS = dermaseptin; DRP = dermaseptin-related peptide; ppdis = preprodistinctin

Genus, species Peptide Abbreviation Sequence Accession number Caerin-related peptide Agalychnis A. annae A1 CRP-A1(DRP-AA-3-4) GMWGSLLKGVATVVKHVLPHALSSQQS O93225 A. callidryas C1 CRP-C1(CRP-AC1) GMWGTVFKGIKTVAKHLLPHVFSSQQS B6HY14 Orphan peptide Agalychnis A. dacnicolor (Pachymedusa dacnicolor) DRP-PD-3-7 LLGDLLGQTSKLVNDLTDTVGSIV O93455 Phyllomedusa P. sauvagii DRS-S9 GLRSKIWLWVLLMIWQESNKFKKM Q1EN15 P. hypochondrialis Distinctin-like (ppdis-H1) NLVSALIEGRKYLKNVLKKLNRLKEKNKAKNSKENN Q17UZ0

TABLE 8. Examples of Neuropeptides and Hormones of the Dermaseptin Superfamily from South American Hylid Frogs (subfamily Phyllomedusinae). X = Leu or Ile. hyP = hydroxyproline. a = C-terminal amidation. HPS = hyposin; PRP = Phe-rich peptide

Genus, species Peptide Sequence Accession number Bradykinin and related peptides Phyllomedusa P. hypochondrialis/azurea Phyllokinin RPPGFSPFRIY(SO3H) P84900 [Thr6, Val10]-phyllokinin RPPGFTPFRVY AM283482 P. sauvagii [Thr6]-phyllokinin RPPGFTPFRIY(SO3H) Q800F1 P. hypochondrialis/azurea [Val1, Thr6]-bradykinin-Leu-Thr VPPGFTPFRLT Q0VTU0 [Thr6]-bradykinin RPPGFTPFR AM283482 [Val1, Hypro2]-bradykinin VhyPPGFTPFR Q0VTT8 P. rohdei Phyllokinin RPPGFSPFRIY Q7LZ52 P. sauvagii [Thr6]-bradykinin RPPGFTPFR Q800F1 Hyposin Phasmahyla P. jandaia HPS-J1 FRPAXIVRTGKa P86613 Phyllomedusa P. hypochondrialis/azurea HPS-HA1 LRPAVIRPKGKa P84954 HPS-HA2 LRPAFIRPKGKa P84955 HPS-HA3 LRPAVIVRTKGKa P84956 HPS-HA4 FRPALIVRTKGTRLa P84957 HPS-HA5 LGPALITRKPLKGKP P84958 P. sauvagii HPS-S1 (sauvatide) LRPAILVRTKa C5J8E3 Tryptophyllin Agalychnis A. dacnicolor (Pachymedusa dacnicolor) PdT-1 KPhyPAWVPa P83455 PdT-2 DMSPPWHa C7C1L9 Phyllomedusa P. hypochondrialis/azurea Pha-T1 RPPSWIPK Q0VKG8 Phe-rich peptide Phyllomedusa P. hypochondrialis/azurea PRP-HA1 FLFFAFHPLa Q0JRV5 Dermorphins/deltorphins Agalychnis A. dacnicolor Dermorphin YAFGYPSa O93456 (Pachymedusa dacnicolor) [Ile2]-deltorphin YIFHLMD O93456 A. annae Dermorphin YAFGYPSa O93227 Phasmahyla P. jandaia Dermorphin YAFGYPSa P86633 Deltorphin YMFHLMDa P86634 Phyllomedusa P. bicolor [D-Ala2]-deltorphin-1 YAFDVVGa P21850 [D-Ala2]-deltorphin-2 YAFEVVGa P21850 [D-Ala2]-dermorphin-like YAFGYP P21850 [D-Ala2]-dermorphin-like YAFWYPN P21850 P. burmeisteri Deltorphin-like peptide YMFHLMNa P86285 P. hypochondrialis/azurea Dermorphin YAFGYhyPSa P85887 P. sauvagii Deltorphin (dermenkephalin) YMFHLMDa P05422 Dermorphin YAFGYPSa P05422

TABLE 9. Canonical Precursor Architecture of Antimicrobial Peptides and Neuropeptides from the Dermaseptin Superfamily. A/Diagram of Preprodermaseptin. The Coding Region, Including the Signal Peptide, the Acidic Propiece and the Peptide Progenitor Sequence is Drawn as a Rectangle. The Signal Peptide Includes the First 22 Amino Acid Residues, while the Acidic Propiece Comprises 19–26 Residues. B/Examples of Amino Acid Sequences of Preprodermaseptins Encoding Antimicrobial Peptides and Neuropeptides from South American (Subfamilies: Phyllomedusinae and Hylinae) and Australian Hylid Frogs (Subfamily: Pelodryadinae), and from North American, Asian, and European Frogs Belonging to the Ranidae and Dicroglossidae families. Identical Amino Acids have shaded backgrounds. Highly Similar Amino Acids are Colored Cyan. Accession number: http://www.uniprot.org/uniprot/Ref

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Biological Actions

The pharmacology of amphibian opiates has been extensively described.5,16,18,22,23

Dermorphin has a high affinity and selectivity for μ-opioid receptors: Its opioid potency in guinea pig ileum (GPI) preparations is 100 times higher than that of morphine. The amidated analogs of Ph. bicolor dermorphins display a similar or slightly higher μ-opioid receptor affinity but a significantly higher (15–20 times) κ-opioid receptor affinity than dermorphin does (Table 2). Like μ-opiate agonists, dermorphins produce antinociception and also catalepsy, respiratory depression, constipation, tolerance, and dependence, although at a lower degree than morphine does. Dermorphin-induced antinociception takes place at both the supraspinal and spinal levels. The analgesic potency of dermorphin is about 280 times higher than that of morphine after intracerebroventricular (icv) injection but comparable to that of morphine after subcutaneous (sc) injection because of its low CNS permeability and bioavailability. [Lys7]dermorphin and some synthetic analogs bearing a hydrophilic group (a basic amino acid or a glycosyl residue) at the C-terminal end of the molecule enter the CNS at a 7–10 times higher rate than dermorphin does, suggesting facilitated transport across the blood brain barrier by a carrier or endocytosis. The amphibian opioid with the highest analgesic potency and efficacy is [Lys7]dermorphin (AD50: 30 pmol per rat, by the icv route; 0.22 μmol/kg, by the sc route). Its antinociceptive effect lasts more than 3 h and is never accompanied by catalepsy. By contrast, [Trp4,Asn7]dermorphin induces catalepsy at icv doses that do not modify the reaction time in the tail-flick test: The ratio of antinociceptive to cataleptic ED50 (AD50/CD50) is 40, a value 500 times higher than that calculated for [Lys7]dermorphin (AD50/CD50 = 0.073). The ratio of antinociceptive to respiratory-depressant ED50 doses is 17 times lower for [Lys7]dermorphin than for morphine. Indeed, [Lys7]dermorphin, in the range of the analgesic doses (36–120 pmol, icv; 0.12–4.7 μmol/kg, sc) significantly increases respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. The early onset ventilatory stimulation produced by [Lys7]dermorphin, and ascribed to the intact peptide, is mediated by serotonergic descending excitatory pathways that stimulate neurons of the brain stem respiratory network.22 In rats and mice, central or peripheral administration of the dermorphin-like peptides induces a significantly slower development of tolerance to the antinociceptive effect than morphine does. Withdrawal symptoms precipitated by naloxone are less intense in peptide-dependent than in morphine-dependent rats. Like morphine, dermorphin inhibits gastric acid secretion, delays gastric emptying, and slows down intestinal and colonic propulsion. Compared with dermorphin, [Lys7]dermorphin is slightly (3–5 times) more potent in both reducing acid gastric secretion and gastric emptying. By contrast [Trp4,Asn7]dermorphin is 20–50 times less potent than dermorphin in inhibiting gastric acid secretion but is 100–1000 times less potent in delaying gastric emptying. In spite of comparable μ-opioid receptor affinity, the two dermorphins from Ph. bicolor, [Lys7]dermorphin and [Trp4,Asn7]dermorphin, differ in biological effects, underling distinct functional roles of the μ-opioid receptors.

TABLE 2. Affinity and Selectivity for μ-, δ-, and κ-Opioid Receptors, Biological Activities on Guinea Pig Ileum (GPI) and Mouse Vas Deferens (MVD) Preparations and Analgesic Potency of Dermorphins and Deltorphins

Compounds Ki, nM (mean ± S.E.) IC50, nM (mean ± S.E.) Analgesia μ δ κ GPI MVD AD50, nmol per rat Morphine 11 ± 1.5 500 ± 48 150 ± 18.5 1215 ± 115 8.7 ± 1.1 Dermorphin dermorphin 0.6 ± 0.02 929 ± 41 8162 ± 979 1.29 ± 0.11 16.5 ± 1.3 0.035 ± 0.01 [Hyp6]der 0.7 ± 0.03 1200 ± 131 – 1.6 ± 0.12 18.1 ± 2.9 – [Lys7]der 0.09 ± 0.008 1105 ± 185 617 ± 66 1.15 ± 0.13 13.6 ± 1.5 0.026 ± 0.009 [Lys7-OH]der 5.7 ± 0.51 1150 ± 172 – 3.82 ± 0.45 56.3 ± 7.8 – [Trp4,Asn5]der 0.9 ± 0.052 480 ± 45 177 ± 12 5.00 ± 0.52 73.7 ± 9.1 0.43 ± 0.04 [Trp4,Asn5-OH]der 4.44 ± 0.39 715 ± 73 2396 ± 301 13.1 ± 1.2 205 ± 25 – [Trp4,Asn7]der 0.32 ± 0.026 690 ± 57 427 ± 47 0.58 ± 0.06 6.6 ± 0.9 2.86 ± 0.31 [Trp4,Asn7-OH]der 2.9 ± 0.2 865 ± 69 905 ± 61 1.3 ± 0.2 10.4 ± 1.3 – Deltorphin D-Ala2-delt I 1985 ± 224 0.78 ± 0.08 >10,000 1239 ± 203 0.18 ± 0.02 15 ± 3.0 D-Ala2-delt II 2222 ± 233 1.03 ± 1.09 >10,000 2500 ± 170 0.37 ± 0.03 54 ± 6.0 D-Met2-delt 693 ± 37 1.18 ± 0.21 >10,000 1476 ± 185 0.97 ± 0.05 20 ± 5.0 D-Leu2-delt >10,000 >10,000 >10,000 >5000 2480 ± 378 – D-Ile2-delt 1021 ± 57 24 ± 3 >10,000 4200 ± 475 7.0 ± 0.9 6.7 ± 1.0 D-aIle2-delt 452 ± 68 54 ± 65 >10,000 3200 ± 307 70 ± 8.2 9.8 ± 1.2

The discovery of deltorphins provided the tools for the functional characterization of the δ-opiate system. Although D-Ala2-deltorphins have δ-binding affinity similar to D-Met2-deltorphin, they consistently have the highest δ-opioid selectivity (Table 2). The rank order of selectivity (Kiδ/Kiμ) is D-Ala2-deltorphin-I = D-Ala2-deltorphin-II > D-Met2-deltorphin > > D-Ile2-deltorphin >> D-Leu2-deltorphin heptadecapeptide or its N-terminal decapeptide fragment. Both D-Met2-deltorphin and D-Ala2-deltorphins are highly resistant to enzyme degradation. D-Ala2-deltorphin-I and -II cross the blood–brain barrier in vivo and in vitro: D-Ala2-deltorphin-II is a transport substrate of organic anion transporting polypeptides (Oatp/OATP), a family of polyspecific membrane transporters, strongly expressed in the rat and human blood–brain barrier.

In mice, D-Ala2-deltorphin II, by the icv route (EC50 = 2.1 nmol per mouse) is half as potent as morphine and the δ-selective antagonist, naltrindole, antagonizes its analgesic effect. Repeated injections of D-Ala2-deltorphin-II induce tolerance to the antinociceptive effect. Isobolographic analysis shows that the supraspinal antinociception induced by the δ-opioid agonist DPDPE and the spinal antinociception induced by D-Ala2-deltorphin-II are synergistic in many nociceptive tests. In rats, intrathecal injections of D-Ala2-deltorphin-II (from 0.6 nmol per rat) produce a naltrindole-reversible inhibition of the tail-flick response by inhibiting the nociceptive neurons in the superficial and deeper dorsal horn of the medulla. Conversely, when injected icv in rats, D-Ala2-deltorphin-II is a weak partial agonist (>30 nmol per rat) and induces fleeting naloxone-sensitive antinociception. Both D-Ala2-deltorphin-I and D-Met2-deltorphin at doses between 6.5 and 52 nmol per rat induce a naloxone-sensitive analgesia.

Results suggest that the δ-agonists play a predominantly modulatory role in antinociception rather than a primary role. In mice and in rats, the intensity of δ-opioid analgesia depends on the coactivation of μ-opiate receptors by endogenous or exogenous opioids. Accordingly, deltorphin-induced analgesia is weaker in homozygote mice with a disrupted μ-opiate receptor gene than in wild-type mice.

Injections of the deltorphins into the rat brain ventricles, ventral tegmental area, and nucleus accumbens at doses 10–100 times lower than those inducing analgesia (0.06–3.8 nmol per rat) invariably increase locomotor activity and induce stereotyped behavior. The δ-selective antagonist, naltrindole, antagonizes deltorphin-induced motor activity. Repeated icv injections of D-Ala2-deltorphin-II in naive rats induce tolerance to the stimulant effects. High doses (10–50 nmol per rat) of all the D-Met2-deltorphin analogs and His4-substituted D-Ala2-deltorphins induce nonopioid motor dysfunction that is completely blocked by the noncompetitive NMDA antagonist, dextrorphan. Deltorphin improves memory consolidation in a passive avoidance test in mice. D-Ala2-deltorphin-I, at low doses, stimulates respiratory activity in fetal lambs. D-Ala2-deltorphin-II inhibits diarrhea induced by castor oil and colonic bead expulsion, but it leaves the rate of transit through the small intestine unchanged. By the sc route, D-Ala2-deltorphin-I inhibits acidified alcohol-induced gastric mucosal lesions. It is involved in the control of ingestive behavior, stimulating food intake and angiotensin-mediated water consumption in rats.

The discovery of the amphibian opioid peptides confirms that the amphibian skin and its secretions offer an inexhaustible supply of biologically active peptides. The amphibian peptides isolated from methanol extracts of amphibian skin are of a small or relatively small molecular mass (700–4600 Da). Proteins, externally secreted by syncytial cells forming the wall of the integument glands, can be obtained upon electrical stimulation of the skin of the living frog. By repetitive electrical stimulation at weekly intervals, several milligrams of bioactive proteins can be collected from a single frog, yielding an amount of biologically active proteins that is enough for pharmacological research. This is the case of the new Bv8/prokineticin family.

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2004, ToxiconWudayagiri Rajendra, ... Kandiah Jeyaseelan

Deltorphins (also referred as dermenkephalin) and related peptides isolated from the frog skin have been found to exhibit high selectivity for δ-opiate receptors (Kreil et al., 1989) and produced analgesia in laboratory animals and humans (Basso et al., 1985; Sandrini et al., 1986). Synthetic analogs of deltorphins such as [nBuG6]-DLT and [iso BuG6]-DLT also exhibited similar anti-nociceptive potency through the mediation of δ-opioid receptors located in the periphery as well as the central nervous system (Chiba et al., 1996). Dermorphin/deltorphin-like peptides and their synthetic analogues, having different degrees of affinities for μ- and δ- opiate receptors, showed potent analgesic effect (Negri et al., 1992; Labuz et al., 2003).

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4.1.5 Other receptors

In addition to the common receptors, some other receptors were also reported to promote the active transport of nanocarriers across the BBB and targeting lesion sites [134]. As initially proposed by Chen et al., the μ-opioid receptor [135] was extensively expressed in the brain cells, and its targeting ligand, dermorphin, was regarded as a promising ligand for the BBB penetration and glioma targeting [136]. Moreover, to promote the efficiency of brain targeting and elevate the precision of MR imaging, Song et al. proposed an acetylated and Cu(II)-complexed dendrimers nanoplatform (M-CSTD.NHAc/Cu(II)) that covalently conjugated with pyridine (Pyr), dermorphin and RGD and spontaneous assembled for MR imaging-guided therapy in orthotopic glioma [137]. In particular, dermorphin was constructed as the first-order ligand to target μ-opioid receptors in the BBB, while RGD was employed as the secondary ligand binding integrins on glioma cells. Compared with the CuCl2 group, M-CSTD.NHAc/Cu(II) group exhibited pronounced Cu uptake by C6 cells. Besides, M-CSTD.NHAc group showed the highest efficiency of cell uptake at the same CSTD concentration (10 μM), indicating that dermorphin facilitated the BBB crossing. It was notable that M-CSTD.NHAc/Cu(II) was capable of T1-weighted MR imaging and displayed significant efficacy of chemodynamic therapy in glioma-bearing mice.

To simultaneously alleviate epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor activation, Meng et al. designed NHS-PEG8-Mal modified poly-2-methacryloyloxyethyl phosphorylcholine-nanocarrier (BIP-MPC-NP), which conjugated with Inherbin3 and cMBP [138]. MPC, a zwitterionic polymer, empowered the nanocarriers with long blood circulation time owing to its remarkable biocompatibilities. Given the dual decoration of EGFR ligand Inherbin3 and mesenchymal-epithelial transition factor ligand cMBP, BIP-MPC-NP exhibited higher uptake by LN229R cells than MPC-NP without peptides attachment. After the tail vein injection, a significant fluorescence signal was measured in LN229R tumor-bearing mice, which showed a 3.2-fold increase in comparison to BIP-NP, indicating the superior penetration into glioma cells. Furthermore, BIP-MPC-NP inhibited the downstream signal of the EGFR and mesenchymal-epithelial transition factor, and mitigated E2F1 expression, resulting in attenuation of DNA damage repairment. Importantly, the combined treatment of temozolomide and BIP-MPC-NP tremendously prolonged the median survival time in the temozolomide-resistant glioma model.

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Amphibian Skin Opioids

The first peptide family of amphibian opioids was discovered in 1981 and named dermorphins. Until the discovery of mammalian endomorphins by Zadina and colleagues, these peptides represented the most potent and selective μ-opioid receptor agonists identified in living organisms. Nine years later, deltorphins were discovered in the amphibian skin; these peptides are still the most potent and selective δ-opioid agonists available today.

A unique characteristic of amphibian opioid peptides (Table 3) is the presence in the second N-terminal position of a d-amino acid residue that confers on these compounds high resistance to enzyme degradation. Amphibian opioids have been found only in the skin of South American hylid frogs belonging to the subfamily Phyllomedusinae (Phyllomedusa, Agalychnis, and Pachymedusa species). Although pharmacologists discovered these opioids in Amazonian frogs comparatively recently, the Matses of the upper Amazonian basin knew of the pharmacological properties of amphibian skin opioids for a long time. For centuries they had habitually applied the dried skin secretions of Phyllomedusa bicolor, called sapo (the Spanish word for toad), to cuts in their skin during shamanistic hunting rituals. The abundance of deltorphins and dermorphins acting together with, or probably synergistically with, the other active peptides present in these secretions might have caused analgesia and behavioral excitation in the hunters.

Origin of the Amphibian Skin Opioids

The d-amino-acid-containing opioid peptides issue from precursors showing a common preproregion (a 22-residue signal peptide and a 18- to 25-residue acidic prosequence) with precursors of the peptide antibiotics dermaseptins (24- to 34-residue polycationic and α-helical amphipathic peptides). Of the three types of dermal glands (mucous, lipid, and granular) in the skin of Phyllomedusa, only the granular glands are specifically involved in the biosynthesis and secretion of dermaseptins and deltorphins. The granular glands are the largest glands in the Phyllomedusa skin; they lie deeper in the epidermis, are lined by epithelium that is more a syncytium, and are surrounded by a layer of myoepithelial cells involved in the holocrine rapid discharge of secretory products collected in roundish granules. The granules do not bud off from the membranes on the Golgi apparatus but seem to be generated in the vacuoles of the vacuolated stage during gland development.

All the amphibian skin opioids contain the N-terminal sequence Tyr-d-Xaa-Phe, where the aromatic residues of Tyrl and Phe3 are of l-configuration and the d-Xaa in the second position of the molecule is a d-amino acid (d-Ala or d-Met). The d-enantiomer is encoded, however, by the codon for the l-isomer in the precursor cDNA. Thus l-Xaa2 must be converted to d-Xaa2 by an unusual posttranslational reaction that presumably takes place in the precursor itself. Because [l-Xaa2]-containing peptides have never been found in amphibian skin extracts, the epimerization mechanism probably involves a quantitative inversion of the chirality of the α-carbon of the amino acid residue rather than a racemization, which would yield an equimolar mixture of l- and d-isomers. Enzymes catalyzing the formation of d-amino acids are known so far only in yeast. From Bombina skin secretions, Kreil and colleagues recently purified a 52 kDa glycoprotein which catalyzes the reaction Ile-Ile-Gly to Ile-d-allo-Ile-Gly. The partial conversion of Ile to d-allo-Ile in peptide linkage proceeds without the addition of cofactors.

Amphibian Opioids and Opioid Receptors

Despite the common N-terminal tripeptide (Tyr-d-Xaa-Phe), the two families of amphibian opioids differ widely in receptor selectivity, although they bind to their own receptors with similar affinities. The N-terminal domain contains the minimum sequence essential for opioid receptor binding, whereas the C-terminal domain contains the address requisites for receptor selectivity. The presence of negatively charged amino acid residues (Asp 114 and Asp 147) that contribute to ligand binding within the putative transmembrane domains II and III of the μ-opioid receptor protein, may explain why positively charged dermorphins have high μ-opioid receptor selectivity and why amidation of their C-terminal group increases their affinity and potency. Similarly, the negatively charged C-terminal tetrapeptide of deltorphins enhances deltorphin selectivity for the δ-opioid receptor by electrostatic attraction to the positively charged binding site of the δ-opioid receptor (Arg 292) and electrostatic repulsion from the negatively charged μ-opioid receptor site. Electrostatic attraction within the peptide molecule may explain why deltorphins have a folded structure, whereas dermorphins have a more distended and flexible structure. Unlike the positively charged C-terminus of dermorphins, the negatively charged C-terminal tail of deltorphins comes into close contact with the positively charged N-terminal tripeptide of the molecule and folds the backbone, thus placing the Tyr1 and Phe3 aromatic rings in definite orientations best suited for δ-opioid receptor docking.

Dermorphin affinity for μ-opioid receptors and its opioid potency are 20 and 100 times higher respectively than those of morphine. Among other dermorphins, [Lys7]dermorphin shows an affinity and selectivity for μ-opioid receptors 6 times higher than dermorphin and 100 times higher than morphine. Like μ-opioid agonists, dermorphins produce not only antinociception but also catalepsy, respiratory depression, constipation, tolerance, and dependence, albeit to a lower degree than morphine. Dermorphin-induced antinociception takes place at the supraspinal and spinal levels. Owing to its low central nervous system (CNS) permeability and bioavailability, the analgesic potency of dermorphin is about 250 times higher than that of morphine after intracerebroventricular (ICV) injection but comparable to that of morphine after subcutaneous (SC) injection. [Lys7]dermorphin and some synthetic analogs bearing a hydrophilic group (a basic amino acid or a glycosyl residue) at the C-terminal end of the molecule enter the CNS in 7–10 times higher amounts than dermorphin, suggesting facilitated transport across the blood–brain barrier by a carrier or endocytosis.

Although d-Ala-deltorphins have δ-opioid binding affinity similar to d-Met-deltorphin, they consistently have the highest δ-opioid selectivity. The rank order of selectivity (Kiδ/Kiμ) is d-Ala-deltorphin-I = d-Ala-deltorphin-II >> d-Met-deltorphin. The high δ-opioid selectivity of d-Ala-deltorphins can be attributed to their C-terminal tetrapeptide sequence, in which the anionic residue plays an important role. Elimination of the charge at the fourth position normally results in opioids that have similar δ- and μ-opioid affinity and generally lack selectivity. Substitution of Gly in the fourth position permits d-Ala-deltorphins to assume a more extended conformation, dramatically increasing their μ-opioid affinity and potency. The hydrophobic qualities of the residues at the fifth and the sixth positions (Val5–Val6) are crucial in maintaining the affinity and selectivity of d-Ala-deltorphins for δ-opioid receptors, as evidenced in peptide analogs in which the aliphatic quality of the side chain was enhanced.

When d-Ala-deltorphins became available, pharmacological and biochemical studies provided evidence of distinct δ1- and δ2-opioid receptor subtypes in the rodent CNS. Studies comparing the binding properties of [3H]deltorphin-I and d-Pen2, d-Pen5enkephalin (DPDPE) in rat brain synaptosomes, provided the first biochemical evidence for two δ-opioid receptor subtypes. In the mouse brain, homogenates and in rat brain slices, d-Ala-deltorphin-II binds preferentially to a population of δ-opioid receptors that develop after weaning and correspond to δ2-opioid receptor subtype. The accumulated evidence demonstrates that d-Ala-deltorphin-II exerts antinociceptive action at δ2-opioid receptor subtype, whereas d-Ala-deltorphin-I (and DPDPE) interact with greater specificity at δ1-opioid receptor subtype. Using selective δ-opioid antagonists, tolerance development, and δ-opioid receptor knockdown (antisense oligonucleotide-treated) mice and rats, numerous pharmacological studies also suggested that spinal and supraspinal antinociception produced by DPDPE/d-Ala-deltorphin-I and d-Ala-deltorphin-II in rats and mice are mediated by distinct δ-opioid receptor subtypes.

Despite pharmacological evidence of two distinct δ-opioid receptors, molecular biologists have not yet succeeded in cloning δ-opioid receptor subtypes. The available evidence implies that the relatively small change in the longer side chain on Glu, due to the methylene C–C bond, could influence the global conformation of d-Ala-deltorphin-II, exerting a role in selecting the postreceptor transduction pathway by differentially activating δ-opioid receptors.

d-Ala-deltorphin-II induces δ-opioid receptor-mediated analgesia in frogs and also in the invertebrate land snail (Cepaea nemoralis). When administered by intrathecal injection in rats, d-Ala-deltorphin-II produces a dose-related inhibition of nociceptive responses. Its inhibitory effect lasts 10–60 min, depending on the dose, and is naltrindole-reversible. Conversely, when injected ICV in rats, d-Ala-deltorphin-II was a weak partial agonist; only doses higher than 30 nmol produced some degree of antinociception and none of the doses elicited the maximum achievable response. Repeated injection of d-Ala-deltorphin-II induces tolerance to the antinociceptive effect. There is no cross-tolerance between antinociception induced by d-Ala-deltorphin-II and that generated either by μ- or by δ1-opioid receptor agonists. The finding that intrathecal injection of d-Ala-deltorphin-II has a higher analgesic effect than DPDPE is probably related more to the predominance of δ2-opioid receptors in the spinal cord than to a prevalence of supraspinal δ1-opioid receptors. Isobolographic analysis revealed that supraspinal/spinal antinociceptive interactions for both the δ1-opioid agonist DPDPE and the δ2-opioid agonist D-Ala-deltorphin-II were synergistic in many nociceptive tests, suggesting that compounds acting through δ-opioid receptors may have sufficient potency for eventual clinical applications.

Data suggest that the δ-opioid agonists play a predominantly modulatory role in antinociception rather than a primary role. In homozygote mice with a disrupted δ-opioid receptor gene, δ-opioid agonist-induced analgesia is reduced. Many studies showed that in mice and in rats the intensity of δ-opioid analgesia depends on coactivation of μ-opioid receptors by endogenous or exogenous opioids. Stress associated with the ICV injection may activate μ-opioid receptors through the release of endogenous opioids and thus potentiate the antinociceptive responses to δ-opioid agonists. Moreover, δ-opioid agonists can be regarded as potential drugs for the treatment of chronic pain: in rats; intrathecal administration of d-Ala-deltorphin-II dose-dependently antagonized the cold-water allodynia which developed after sciatic nerve injury, and ICV administration of d-Ala-deltorphin-II significantly reversed the hyperalgesic response associated with peripheral inflammation.

Injections of d-Ala-deltorphin-II into the rat brain ventricles, ventral tegmental area, and nucleus accumbens invariably increase locomotor activity and induce stereotyped behavior. The ambulatory activity is intermittent and usually intercalated by rearing events. The motor activity is antagonized by the δ-opioid-selective antagonist naltrindole, and by high doses of the μ-opioid antagonist naloxone, but is unaffected by the δ1-opioid-selective antagonist naloxonazine. ICV administration of 1.3 nmol/rat of d-Ala-deltorphin-II increases social contacts in rats. Local application of d-Ala-deltorphin-II to the nucleus accumbens, but not to the nucleus caudatus, increases extracellular dopamine concentrations (by up to 120%). It also stimulates locomotor activity and stereotypes. Repeated ICV injection of d-Ala-deltorphin-II in naive rats induces tolerance to the stimulant effects, whereas repeated daily injections or continuous infusion of morphine results in sensitization to the behavioral-activating effects of the δ-opioid agonist. Deltorphin improves memory consolidation in a passive avoidance apparatus in mice; this effect is abolished by naltrindole. d-Ala-deltorphin-II causes hypothermia in cold-adapted animals. In contrast to μ-opioid agonists, d-Ala-deltorphin-I, at low doses, stimulates respiratory activity in fetal lambs, and this effect is blocked by the simultaneous administration of naltrindole.

The discovery of the amphibian opioid peptides, apart from the intriguing problem of the occurrence of their analogs in the mammalian central and peripheral nervous systems, has provided new insights into the functional role of the μ- and δ-opioid systems.

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4.1 Targets for peptide-decorated nanocarriers

Various macromolecules and proteins, such as low-density lipoproteins, nicotinic acetylcholine and transferrin, rely on RMT for transcytosis across the BBB through binding to specific receptors on the surface of brain ECs. Hence, peptide-decorated nanocarriers have been explored for brain delivery across the BBB via RMT in several brain diseases, such as glioblastoma, PD and seizure disease. In this section, we illustrate these peptide-modified nanocarriers classified by their targeting receptors. Notably, receptor-targeted delivery strategies may play double-edged roles in drug delivery. Even though the drug delivery systems are designed based on differential expression of receptors in specific tissues, they may still lack the ability to distinguish different tissues in vivo. While the drug accumulation in the lesion is enhanced and their residence time is increased, their distribution in other tissues such as the liver, spleen or kidneys, may also be greater than that of the non-targeted delivery systems. Therefore, more attention should be paid to the distribution of nanoparticles in non-target sites and their impacts on disease processes.

4.1.1 Integrins

Integrins are recognized as a family of adhesion molecules and transmembrane receptors mediating bidirectional signal transduction, consequently regulating cell adhesion, migration and growth [83,84]. Moreover, integrins are involved in immune responses, leukocyte traffic and development [85,86]. As a class of import receptors, integrins are widely distributed in normal cells, such as epithelial cells, smooth muscle cells, osteoclasts and ECs [87]. Under disease conditions such as glioma, integrins exhibit high expression levels in tumor tissues since they are hijacked as tumor nourish signals, increasing tumor invasion and metastasis potentials [88,89]. Thus, integrins are considered as a prospective target for glioma targeted delivery of imaging agents and therapeutics owing to the high expression on both brain capillary ECs and glioma cells [90].

Arg-Gly-Asp (termed RGD), a readily synthesized tripeptide motif with a great affinity towards integrin receptors and low toxicity, has been widely explored as a targeting ligand for brain delivery [91,92]. For example, Lu et al. synthesized drug-loaded nanocrystals based on c(RGDyK) camouflaged with red blood cell (RBC) membrane for preferential drug accumulation at tumor sites (Fig. 3A) [93]. Without drug loading, c(RGDyK) modified RBC vesicles (RGD-RVs) showed strengthened binding affinity towards integrin αVβ3 compared to the unmodified RVs. After loading with docetaxel (DTX), RGD-RBC-NC(DTX) showed superior tumor accumulation, significant tumor growth inhibition and remarkable survival prolongation in the orthotropic glioma mice model (Fig. 3A). Most importantly, RGD-RBC-NC(DTX) prominently prolonged median survivals of mice bearing orthotopic glioma, whereas RBC-NC(DTX) displayed little therapeutic efficacy due to the existence of BBTB, elucidating that RGD facilitated the precise targeting to glioma and effective traverse over the BBTB via RMT.

Fig. 3. Targeting peptide-decorated nanocarriers for brain-targeted delivery. (A) Schematic depicting the preparation of c(RGDyK)-modified cell membrane-coated drug nanocrystals. Kaplan-Meier survival curves of orthotropic glioma bearing receiving different nanocrystals. Median survivals: PBS group = 32 d, TXT group = 32.5 d, RBC-NC(DTX) group = 34.5 d, RGD-RBC-NC(DTX) group = 62 d, Adapted with permission from Ref. [93]. Copyright 2019 American Chemical Society (B) Schematic depicting the aggregation of gold nanoparticles (Au–AZ and Au–AK) in tumor acidic interstitium. Blood-brain barrier permeable nanoparticle aggregates are trapped in tumor tissue. Adapted with permission from Ref. [105]. Copyright 2017 John Wiley & Sons. (C) Design of an electric-field-responsive contrast agent consisting of a paramagnetic polymer encapsulating a superparamagnetic cluster of ultrasmall superparamagnetic iron oxide. Adapted with permission from Ref. [108]. Copyright 2020 Springer Nature.

To precisely delineate brain glioma margins, Liu et al. synthesized RGD-modified Hepatitis B virus core protein (RGD-HBC) to encapsulate organic molecules A1094 [94]. Compared with A1094@HBc, the human glioblastoma cell line (U87) showed higher cellular uptake to A1094@RGD‐HBc, suggesting the excellent targeting ability of RGD towards glioblastoma cells. After 2 h post‐injection, A1094@RGD‐HBc resulted in a tremendously strong near-infrared fluorescence signal in the glioma-bearing mice, whereas no sufficient signal was observed in the normal groups, indicating their superior tissue-specific selectivity. Moreover, the brain tumor imaging of mice after A1094@RGD‐HBc treatment exhibited enhanced photoacoustic signal in photoacoustic imaging and elevated radioactive uptake in microSPECT/CT image. Similarly, Gao et al. constructed a dual-targeting nanoplatform Den-RGD-Reg, which modified G5 dendrimer with both c(RGDyK) and regadenoson (Reg), an adenosine 2A receptor agonist [95]. Firstly, c(RGDyK) targeted integrin αVβ3 on the surface of glioma neovasculature, and then Den-RGD-Reg compromised TJs in BBB via adenosine 2A receptor signaling triggered by the interaction of Reg and adenosine 2A receptor. Compared with paclitaxel (PTX) alone, Den-RGD-Reg prolonged the survival rate of U87-bearing mice via T2W-MR image-guided administration of PTX. Besides, Kuthala et al. synthesized 10B-enriched boron nanoparticles for boron neutron capture therapy (BNCT) towards murine ALTS1C1 glioma models. The surface of these nanoparticles was decorated with RGD-K peptide, which could help the accumulation of boron atoms in the tumor cells via receptor-mediated endocytosis. Accordingly, the nanoparticles could achieve dual-function with both MR imaging and BNCT. Despite achieving noninvasive diagnostic T1-MR imaging of mice brains, these colocalized nanoparticles could also suppress the murine brain tumors after receiving neutron radiation. The mice treated with BNCT nano-medicine exhibited a longer median survival time than other groups [96].

4.1.2 Low-density lipoprotein receptor

Low-density lipoprotein receptors (LDLRs) have been reported as a class of multifunctional cell surface glycoproteins to regulate low-density lipoprotein cholesterol levels [97]. As an important receptor in cell stability, LDLRs are widely distributed in nearly all human organs and tissues, especially adrenals and the liver, which are the main organs for LDL uptake [98]. Besides, they can also be observed in many malignant tumors including glioma cells due to the enormous nutrition demand of highly proliferating cells [99]. In recent years, LDLRs have been gradually identified as a receptor family including a range of related receptors that specifically recognize lipoproteins, such as LDLR-related protein 1 (LRP-1), LRP-2 and scavenger receptor class B type 1 [100]. Among those, LRP-1 attracts much attention owing to its dual-targeting ability towards BBB and glioma cells [101–103].

Angiopep-2, a peptide derived from aprotinin with the Kunitz domains of human proteins, has been regarded as an excellent targeting ligand towards LRP-1 [104]. For example, we prepared a pair of AuNS-based nanoparticles modified by angiopep-2 to guide brain tumor surgery (Fig. 3B) [105]. The angiopep-2 modification enabled the nanoparticles to accumulate at glioblastomas. With the stimulation of the acidic tumor fluid, the nanoparticles initiated the specific assembly and displayed enhanced surface-enhanced resonance Raman spectroscopy signals and magnetic resonance (MR) signals (Fig. 3B). Moreover, superparamagnetic iron oxide (SPIO) is a kind of magnetic nanoparticles that can not only be guided to target tumor sites under magnetic fields but also be employed as MRI contrast agents [106]. Qiao et al. developed an immunochemotherapy method for glioma therapy based on poly[(2-acryloyl)ethyl(p-boronic acid benzyl) diethylammonium bromide] (BA-PDEAEA, BAP) encapsulating superparamagnetic iron nanocubes (SPIONs) [107]. The BAP also contains siRNA against tumor growth factor β (siTGF-β). Besides, it was further coated with temozolomide and zwitterionic-based lipid envelope and finally modified by angiopep-2. In the glioma model, this nanosystem could accumulate via the dual-targeting of angipep-2, leading to the reactive oxygen species-responsive RNA release together with temozolomide and dual cytotoxicities induced by temozolomide and gene silencing efficiency of siTGF-β. Besides, the released SPIONs could be utilized to trace the system using MRI. Furthermore, Wang et al. modified angiopep-2 onto an electrically responsive hybrid micelle, which consisted of paramagnetic poly(ethylene glycol)–poly(lactic acid) (PEG-PLA) copolymer and ultrasmall superparamagnetic iron oxide (USPIO) for seizure treatment in mouse model (Fig. 3C) [108]. Since the angiopep-2 could target LRP-1 on the surface of vascular ECs of the epileptic lesion, the micelle could cross BBB and accumulate at the epileptic region via LRP-1 mediated transcytosis. The seizures associated electric-field could trigger the disruption of the contrast agents, restoring the T1-weighted MR signal quenched by distance with high sensitivity of lesion visualization and improved target-to-background ratio.

4.1.3 Nicotinic acetylcholine receptor

Nicotinic acetylcholine receptor (nAChR) is identified as the first neurotransmitter receptor, accompanied by the initial discovery of neurotransmitter acetylcholine[109,110]. nAChR is a family group expressed in many tissues and cells, such as skeletal muscle, leukocytes, spinal ganglia and central neurons [111]. Among them, α7 nAChR has been pursued as a prominent targeting receptor for brain disease diagnosis and treatment owing to their elevated expression in numerous neurological disorders ranging from PD, senile dementia, and glioma [112,113]. For example, DCDX is a 16-amino acid D-peptide derived from snake neurotoxin [114]. Zheng et al. utilized DCDX for triple-targeting α7 nAChR on glioma cells, glioma vessel endothelium and tumor-associated macrophages [115]. They created a ‘three-birds-one-stone’ delivery strategy for combinatory therapy of glioma by synthesizing a honokiol and disulfiram/copper codelivery liposomal system decorated with DCDX(CDX-LIPO). Consequently, these multiple targeting systems could not only effectively induce the death of glioma cells and activate the tumor-infiltrating macrophage, but also promote M1-macrophage polarization and remodeling of tumor metabolism and immune microenvironment.

Besides DCDX, a 29 amino-acid peptide derived from rabies virus glycoprotein (RVG29) is an elegant option for brain targeted delivery of imaging agents and therapeutic cargoes via nAChR mediation [116–118], as evidenced in traumatic brain injuries[119] and glioblastoma-bearing mice [120]. For example, Liu et al. proposed an RVG2 modified exosome (EXO) coating gene-chem nanocarrier (REXO-C/ANP/S) to accurately deliver the complex of siRNA targeting SNCA and curcumin (C/ANP/S) for PD treatment in MPTP induced PD mice model [121]. With the application of REXO, the therapeutics in core nanocomplex C/ANP/S obtained increased accumulation in bEnd.3 cells and superior migration through epithelial cells in a transwell assay. Besides, RVG contributed to at least 48 h of drug accumulation in mice brains, further improving neuronal recovery and clearance of α-synuclein aggregates in the PD mice model.

Additionally, You et al. conjugated RVG29 to mPEG-PLGA nanocarrier (RNP) for the delivery of deferoxamine (DFO) in PD mice (Fig. 4A) [122]. RVG29 decorated nanoparticles (RNP-DFO) exhibited elevated permeability and prolonged circulation of DFO (3-fold difference) after 6 h post-treatment in the brain tissue by the high binding affinity between RVG29 and its receptor on the brain tissue compared to nanocarriers without RVG29 decoration (NP-DFO). Moreover, they conjugated RVG29 with gold nanoparticles to investigate the critical role of RVG29 in intracerebral delivery. The inductively coupled plasma mass spectrometry results corroborated that RVG29 significantly promoted nanoparticle accumulation in the brain rather than retention in microvessels or ECs. At 6 h after intravenous injection, both NP-Cy5.5 and RNP-Cy5.5 accumulated in the main damaged regions, including the striatum and substantia nigra.

Fig. 4. Nicotinic acetylcholine/transferrin receptor-mediated blood-brain barrier penetration of nanocarriers. (A) Internalization of RVG29-modified nanoparticles (RNP) delivering iron chelators for Parkinson’s disease treatment via nicotinic acetylcholine receptor-mediated transcytosis. Ex vivo fluorescence images of the brain and confocal images of striatum from healthy mice receiving RNP by intravenous injection. Adapted with permission from Ref. [122]. Copyright © 2018 American Chemical Society. (B) Schematic diagram of H-ferritin (HFn) nanocarriers crossing the blood-brain barrier via transferrin receptor-mediated transcytosis. In vivo bioluminescence images of U87 orthotopic tumor-bearing mice after being intravenously injected with different formulations. Adapted with permission from Ref. [128]. Copyright © 2018 American Chemical Society.

4.1.4 Transferrin receptor

TfR is also an important targeting receptor across the BBB [123,124]. There are two types of reported TfRs, TfR1 and TfR2, of which TfR1 is expressed on the surface of many cells, while TfR2 is mainly expressed in iron metabolism-regulating tissues. The TfR has multiple ligands including transferrin (Tf) and lactoferrin [125]. For BBB targeting, Qu et al. utilized the natural brain targeting ability of blood exosomes and loaded dopamine into blood exosomes via a saturated solution incubation method. These dopamine-loaded exosomes could efficiently achieve the striatum and substantia nigra in the PD mouse model. The mechanism of BBB penetration was confirmed as the interaction between natural Tf on exosomes and TfR on BBB. Besides, the intravenously administered dopamine-loaded exosomes exhibited lower systemic toxicity than free dopamine [126].

However, the application of exogenous Tf and their mimetic peptide as transporting cargo crossing BBB is limited, owing to the competitive inhibition for TfR by the endogenous Tf with high levels. To overcome this challenge, a major strategy is to find other specific ligands towards TfR with distinct binding sites from that of the endogenous Tf.

Human heavy chain ferritin (HFn) has been proven to specifically bind TfR1 via different epitopes [127]. To explore whether HFn possessed the capability of transporting the BBB and targeting glioma, Fan et al. attached fluorescent IRdye800 to HFn and measured increased fluorescence signal both in the brain of normal mice and specific tumor area of glioma-bearing mice, whereas the human light chain ferritin (LFn) showed no evident signal (Fig. 4B) [128]. Moreover, the signal margins of IRdye800-HFn corresponded to U87 tumor cells and the accumulation of HFn-decorated nanocarriers in the tumor area was 10-fold higher than that in the healthy tissue. After doxorubicin (Dox) loading, HFn-Dox significantly decreased the weight loss and prolonged the survival time of U87-bearing mice in contrast to free Dox and undecorated liposomal Dox.

HAIYPRH (T7) peptide is another specific TfR ligand and exhibits promising potential for diagnostic and therapeutic delivery system owing to easy synthesis, great stability and distinct from the transferrin binding site [129,130]. For example, Cai et al. designed T7 peptide modified nanocarriers via an acid-cleavable long-chain polyethylene glycol (PEG) to deliver siRNA and D-amino acid inhibitor for AD [131]. Interestingly, the acid-sensitive T7 peptide as the first targeting ligand could be detached from nanocarriers, allowing for further intracephalic drug delivery after endocytosis. T7 peptide modified nanocarriers were labeled with Cy5.5 to monitor the biodistribution in AD model mice. The quantitative fluorescence showed that the brain accumulation of acid-cleavable T7 peptide-modified nanocarriers increased to 1.83 times and that of acid-uncleavable T7 peptide-modified nanocarriers increased to 1.64 times compared with the control group. The results indicated that acid-cleavable T7 peptide could not only bind TfR selectively but also separate quickly from the BBB after endocytosis, facilitating brain drug delivery.

Kim et al. designed T7 peptide-decorated exosome (T7-exo) and demonstrated their efficient capacity of loading antisense miRNA oligonucleotides against miR-21 (AMO-21) into glioma [132]. AMO-21 can be used to efficiently reduce glioma by inhibiting oncogenic microRNA-21 [133]. As aforementioned, the expression of TfR was higher in glioma cells than in normal cerebral ECs. They also designed the RVG-decorated exosome (RVG-exo) as the control group to further investigate the AMO-21 delivery efficiency of T7-exo. After the caudal vein injection, T7-exo exhibited the highest concentration in mice brains in contrast to other control groups, as evidenced by fluorescence intensity in live mice and brain samples. Moreover, both RVG-exo/AMO-21 and T7-exo/AMO-21 groups showed more than 50% decrease in tumor size than that in the control group, whereas the anti-tumor effect was more remarkable in T7-exo/AMO-21 groups. Tumor size was slightly diminished in the Unmod-exo/AMO-21 group to approximately 80%. They also revealed that a low concentration of AMO-21 (42 ng) could result in glioma tumor size reduction.

4.1.5 Other receptors

In addition to the common receptors, some other receptors were also reported to promote the active transport of nanocarriers across the BBB and targeting lesion sites [134]. As initially proposed by Chen et al., the μ-opioid receptor [135] was extensively expressed in the brain cells, and its targeting ligand, dermorphin, was regarded as a promising ligand for the BBB penetration and glioma targeting [136]. Moreover, to promote the efficiency of brain targeting and elevate the precision of MR imaging, Song et al. proposed an acetylated and Cu(II)-complexed dendrimers nanoplatform (M-CSTD.NHAc/Cu(II)) that covalently conjugated with pyridine (Pyr), dermorphin and RGD and spontaneous assembled for MR imaging-guided therapy in orthotopic glioma [137]. In particular, dermorphin was constructed as the first-order ligand to target μ-opioid receptors in the BBB, while RGD was employed as the secondary ligand binding integrins on glioma cells. Compared with the CuCl2 group, M-CSTD.NHAc/Cu(II) group exhibited pronounced Cu uptake by C6 cells. Besides, M-CSTD.NHAc group showed the highest efficiency of cell uptake at the same CSTD concentration (10 μM), indicating that dermorphin facilitated the BBB crossing. It was notable that M-CSTD.NHAc/Cu(II) was capable of T1-weighted MR imaging and displayed significant efficacy of chemodynamic therapy in glioma-bearing mice.

To simultaneously alleviate epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor activation, Meng et al. designed NHS-PEG8-Mal modified poly-2-methacryloyloxyethyl phosphorylcholine-nanocarrier (BIP-MPC-NP), which conjugated with Inherbin3 and cMBP [138]. MPC, a zwitterionic polymer, empowered the nanocarriers with long blood circulation time owing to its remarkable biocompatibilities. Given the dual decoration of EGFR ligand Inherbin3 and mesenchymal-epithelial transition factor ligand cMBP, BIP-MPC-NP exhibited higher uptake by LN229R cells than MPC-NP without peptides attachment. After the tail vein injection, a significant fluorescence signal was measured in LN229R tumor-bearing mice, which showed a 3.2-fold increase in comparison to BIP-NP, indicating the superior penetration into glioma cells. Furthermore, BIP-MPC-NP inhibited the downstream signal of the EGFR and mesenchymal-epithelial transition factor, and mitigated E2F1 expression, resulting in attenuation of DNA damage repairment. Importantly, the combined treatment of temozolomide and BIP-MPC-NP tremendously prolonged the median survival time in the temozolomide-resistant glioma model.

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4 Subcutaneous administration

Subcutaneous tissue is a layer of loose connective tissue and elastin located immediately beneath the dermis and is the main tissue for the storage of fat. And it is distributed with a dense network of capillary rings and a large number of lymphatic vessels [137]. The presence of lipids endows it with a storage effect for vaccines and injected analgesics, thus maintaining their concentration in the tissue for a prolonged period of time, and abundant vascular tissues and lymphatic vessels facilitate the recruitment of immune cells. Additionally, fewer degrading enzymes in the hypodermis guarantee the stability of DDSs. Hence, the hypodermis is an ideal administration site for the sustained release of vaccines and analgesics.

4.1 Administration of opioid analgesics

Opioid analgesics are considered the most helpful therapeutic agents for patients suffering from chronic pain, although they frequently show severe side effects such as gastrointestinal effects (constipation, nausea, and vomiting), respiratory depression, addiction, and abuse potential [138]. Prescriptions for oral medication represent the most accessible mode of administration with minimal invasiveness. However, the first-pass metabolism impairs the drug effect during prolonged treatment regimens. Based on these considerations, Ballet and co-workers developed peptide-based hydrogels to extend the release of opioids via subcutaneous administration. Specifically, they designed and synthesized H-Phe-Glu-Phe-Gln-Phe-Lys-NH2 (P1) based on a previously reported sequence MBG-1, replacing the carboxylic acid group with a carboxamide [139]. This ingenious design increased the gelation pH from 3.49 to 4.02, reducing the occurrence of pain after injection, while maintaining similar gelation properties. Morphine was encapsulated in the hydrogel with a high loading capacity of 77%, which allowed the maintenance of a significant antinociceptive effect with approximately 40% of Maximum Possible Effect after one day, inducing no significant change in general behaviors of mice among therapy process. The all-D sequence corresponding to P1 showed a remarkable increase in resistance toward proteolytic degradation but was accompanied by unexpected cytotoxicity. More interestingly, they considered several structural modifications of P1 to advance the effectiveness of their extended-release platforms [140]. Among all five types of derivatives, P2 (H-Phe-Gln-Phe-Gln-Phe-Lys-NH2), obtained by replacing negatively charged amino acid (Glu) with a polar neutral one (Gln), and P3 (H-Trp-Gln-Trp-Gln-Trp-Lys-NH2) obtained by replacing Phe with Trp, exhibited enhanced effects on long-term drug release. Administrating morphine with P2 or P3 in combination resulted in the sustained, significant painkilling effect at 72 h for P2 or 96 h for P3. In additional studies, they utilized these scaffolds as an extended-release system for the opioid peptide dermorphin, resulting in approximately 1000 times higher potency in human bodies compared to morphine, with fewer side effects as well as longer analgesia [141]. After subcutaneous administration in mice, two different formulations afforded lengthened and significant analgesic effect for 72 h maximum, with one encapsulating drugs within the hydrogels and the other integrating drugs with hydrogelators, denoted as a biogel (Fig. 5A). Furthermore, they determined the structures of the three major metabolites of one biogel in human plasma, corresponding to hepta-, penta-, and tripeptides. Surprisingly, the tripeptidic metabolite exhibited an ultrapotent and long-acting action up to 8 h (Fig. 5B). Their results demonstrated the significance of the gelator sequence biodegradability in drug release behavior. Many other biologically active sequences conjugated to hydrogelators could become promising prodrugs when existing in a controlled-release manner.

Fig. 5. (A) Illustration of self-assembling of supramolecular hydrogels based on amphipathic peptide sequences. The “coformulation” and “biogel” strategies are depicted. (B) In vitro degradation profile of H-Dmt-DArg-Phe-Phe-Gln-Phe-Gln-Phe-Lys-NH2 (6) (in solution) in human plasma. Antinociceptive activity of 6 and its metabolites 17–19 (Met-1 to −3, respectively) in the tail-flick test after subcutaneous administration in mice. Groups of mice received solutions of 6 (3.0 μmol/kg), 17 (3.9 μmol/kg), 18 (4.2 μmol/kg), 19 (6.8 μmol/kg), or saline (control) (right panel). Antinociceptive response, as %MPE, was measured over time, and area-under-the-curve (AUC) values for the respective time curves were calculated. Data are the mean ± standard error of the mean (SEM) of 6–8 mice per group: (*) P < 0.05 vs respective control groups, (#) P < 0.05 vs 6 group, two-way ANOVA followed by Bonferroni post hoc test. MPE, maximum possible effect [141].

4.2 Administration of vaccines

Vaccines have been widely applied to prevent and treat diseases and become a common medical treatment in our daily life [142,143]. Compared with attenuated live vaccines, the subunit vaccines have gained increasing attention due to the definite molecular structures, safety, and antigenicity. Nevertheless, they mostly need to function with the assistance of adjuvants because of their poor immunogenicity due to fast cleanup and low transport effectiveness to the objective in the host [144]. Peptide-based self-assembling hydrogels have been proved to be promising vaccine adjuvants thanks to their excellent biocompatibility, being liable to incorporate and present antigen, and high effectiveness in activating immune responses [145,146].

Collier’s group conducted pioneering studies on self-assembling peptides to improve the output of specific antibodies against subunit vaccines [147]. By employing the Q11 (Ac-QQKFQFQFEQQ-Am) peptide capable of self-assembling into numerous nanofibers with the secondary structure of β-sheet in saline solution, they constructed a peptide O-Q11, which comprised this self-assembling domain and model antigen ovalbumin (OVA) in tandem model (Fig. 6A). O-Q11 elicited an extremely high IgG production in the absence of extra adjuvant in mouse subcutaneous immunization experiments, while utilizing sole fibrillized Q11 didn’t demonstrate any measurable IgG, whether administrated with complete Freund’s adjuvant (CFA) or not. The result indicated that Q11 was not immunogenic. Furthermore, this strong antibody response was completely dependent upon covalently conjugating the antigen and fibrillating domain since no measurable IgG was monitored when mixed OVA and Q11 without covalent conjugation. Furthermore, interferon (IFN)-γ, interleukin (IL)-2, and IL-4 couldn’t be detected in the culture of peptide-challenged splenocytes from the immunologically stimulated mouse, suggesting that the antibody responses didn’t concern obvious induction by T cells. Their strategy represented an easy, chemically defined method of markedly augmenting the antibody response to specific peptide epitope. In addition to short peptide antigens, they creatively incorporated manifold functional βTail (MALKVELEKLKSELVVLHSELHKLKSEL) fusion proteins into self-assembling peptide nanofibers too [148]. The βTail fusion proteins were able to be integrated into fibrillized Q11 solely or in combination at predictable, smoothly graded concentrations, representing an easy but universal means to accurately incorporate combinations of proteins into nano scaffolds (Fig. 6C). Adjusting the dose of antigens to regulate the antibody response was one of the powerful functions of this βTail system constructed by modularity. After immunized by fibrillated Q11 carrying microgram doses of βTail-GFP (green fluorescent protein) or βTail-cutinase, GFP-reactive antibody titers or cutinase-reactive antibody titers elicited by C57BL/6 mice were higher than those immunized with a same dosage of each protein alone. More prominently, they provided concurrent immunity against diverse pathogens and diverse distinct antigens from a unitary pathogen, and minimized immunization times needed, which is of great importance to be a clinical remedy. Furthermore, based on the Q11 peptide and different epitopes, including the universal CD4+ T-cell epitope (PADRE), B-cell epitope from Staphylococcus aureus at specific concentrations, cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), B-cell epitopes from IL-17, and the model CD8+ T-cell epitope SIINFEKL [149–153], they constructed a series of vaccines, making a substantial contribution to the advance of developed immunotherapies and self-assembling peptide hydrogels as adjuvants.

Fig. 6. (A) Illustration of O-Q11peptides self-assembling into nanofibers. (B) IgG titers elicited by OVA, Q11, O-Q11, or OVA + Q11 in PBS or CFA [147]. (C) Schematic of fusion proteins with a β-sheet fibrillizing domain co-assemble with β-sheet fibrillizing peptide Q11 into nanofibers. (D) C57BL/6 mice immunized with Q11 nanofibers bearing βTail-GFP or βTail-cutinase elicit higher titers of anti-GFP or anti-cutinase antibodies, respectively, than mice immunized with βT ail-GFP in PBS [148].

Employing the tetrapeptide GFFY, Yang et al. developed a series of excellent adjuvants that can be used for human immunodeficiency virus (HIV) DNA vaccine delivery or tumor treatment. Their GFFY series of hydrogels include Nap-GFFpY-NMe [154], Nap-GDFDFDpY-OMe [155], Nap-GDFDFDY [156], Fbp-GDFDFDY and Car-GDFDFDY (Fbp = flurbiprofen, Car = carprofen) [157], Nap-GDFDFDYDK [158], Ada-GDFDFDYGDKDKDK-NH2 (Ada = 1-Adamantaneacetic acid) [159], and Fbp-GDFDFDYDK (γE)2-NH2 [160]. These adjuvants are easy to use as they can physically mix with antigens, avoiding a cumbersome preparation process that conjugates the protein or epitope covalently with self-assembling peptides. Nap-GFFpY-NMe and Nap-GDFDFDpY-OMe formed hydrogels via EISA that elicit no detectable antigen-specific immune response. Nap-GFFpY-NMe nanovectors condensed HIV Env DNA, promoted their transfection, and enhanced gene expression in vitro. Furthermore, it strongly activated both humoral and cellular immune responses to a balanced level when applied to mice in vivo, thus effectively improving the prevention and treatment of HIV. Nap-GDFDFDpY-OMe co-assembled with OVA successfully, which increased the IgG production of OVA by 3.8-fold compared with Alum adjuvant used in clinical. However, its L-counterpart only increased the IgG production by 1.3-fold. Further mechanistic studies indicated that the D-peptide hydrogel adjuvant was able to boost antigen uptake, induce DC maturation, facilitate and extend antigen amassing inside lymph nodes, as well as induce germinal center formation. Additionally, the authors demonstrated its antitumor efficacy in an EG-7-OVA tumor-bearing mouse model. Considering challenges in the large-scale synthesis of phosphorylated precursors and side effects caused by EISA (e.g., reproducibility problems and the composition variation), they developed four hydrogels named Nap-GDFDFDY, Fbp-GDFDFDY, Car-GDFDFDY, and Nap-GDFDFDYDK, which could form hydrogels via the heating–cooling process. All obtained hydrogels exhibited great potential as adjuvants to treat cancer using OVA as a model antigen. Further studies indicated that the hydrogel could inhibit cancer by enhancing the uptake of OVA by primary APCs, activating CD8+IFN-γ+T cells, and reducing tumor-associated inflammation. Furthermore, they designed Ada-GDFDFDYGDKDKDK-NH2 self-assembling into nanofibers at a physiological pH. The formed hydrogel could directly capture the protein vaccine through charge interaction between three lysines of the nanofiber and negatively charged protein. As a neo-adjuvant, it was administered subcutaneously, synchronizing the cross-presentation of exogenous antigens as well as innate immune stimulation, inducing the enhanced expression of CD80 and CD86, production of IL-6 and IL-12, and cross-presentation of the SIINFEKL epitope by DCs, in a cooperative manner with NF-κB activation. They recently reported another self-assembling peptide, Fbp-GDFDFDYDK(γE)2-NH2, which served as a supramolecular protein chaperone for OVA and hepatitis B surface antigen. Compared with the previously discussed adjuvants, Fbp-modified D-peptide could influence the COX-2 (cyclooxygenase-2)/PGE2 (prostaglandin) pathway in TME, which reduced immune suppression, in synergy with a strong cellular immune response stimulated by protein antigen, thus significantly retarding tumor growth. Their strategies showed great promise in developing vaccines for tumor therapy and could be applied to other important vaccines.

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URL: https://www.sciencedirect.com/science/article/pii/S0169409X21001745

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